CN1387845A - Medicinal composition for treating autoimmune disease - Google Patents

Abstract

The medicine composition for treating autoimmune diseases contains fluothiamine derivative or its salt in the amount for effective treatment and pharmaceutically acceptable supplementary material. It is one new kind of immunoregulator with inhibition to cellular immunity and humoral immunity. It is proved that the medicine composition has obvious curative effect and low toxicity to autoimmune diseases, including rheumatic arthritis, systemic lupus erythematosus, lupous nephritis, nephropathy syndrome, ankylosing spondylitis, psoriasis and other diseases.

Description

The pharmaceutical composition of treatment autoimmune disease

Technical field

The present invention relates to the treatment of autoimmune disease, be specifically related to Fusaimi medicine composite for curing autoimmune disease.

Background technology

Autoimmune disease is the morbid state that self component generation immunoreation is caused because of body immune system.Its basic feature is: can detect autoantibody and/or self responsiveness T cell that height is tired; Autoantibody or reply the T cytosis in autologous tissue's cell causes damage or dysfunction; Reproduciblely go out similar animal model, can make the disease passive transfer with patients serum or primed lymphocyte; The state of an illness lapses to autoimmune response intensity closely related; The outbreak repeatedly of disease and chronic animal migration; And genetic predisposition arranged.

Just, therefore need carry out long-term treatment and monitoring because autoimmune disease is chronic, outbreak repeatedly.At present normal what adopt is the inflammation that causes of control autoimmune disease and the medicine that suppresses immune response, for example adopts immunosuppressant such as glucocorticoid, alkylating agent, antimetabolite.These medicines comprise prednisone, methotrexate, cyclophosphamide, azathioprine, ciclosporin A etc.But they also suppress immune anti-infectious function and can cause serious adverse effects when suppressing inflammatory reaction that autoimmune response causes and tissue injury.

1981, U.S. scientist Kammerer synthesized leflunomide (leflunomide), was used for the treatment of rheumatoid arthritis by drugs approved by FDA in 1998.Nineteen ninety present inventor and his U.S. tutor Dr.Williams, J.W. carrying out leflunomide inhibition nf allograft skin, kidney, heart, small intestinal and closely be in the animal experiment of rejection of heart xenograft, the metabolite Fusaimi (flucyamide) of leflunomide has been carried out tests such as dynamic metabolism, long term toxicity.The inventor further carries out researchs such as synthesis technique, pharmacodynamics, experimental therapeutic and clinical treatment, thereby has finished the present invention on the basis of these researchs.

Summary of the invention

The object of the present invention is to provide a kind of low toxicity, effectively treat the pharmaceutical composition of autoimmune disease.

Another object of the present invention is to provide the application of this pharmaceutical composition in the pharmaceutical preparation of preparation treatment autoimmune disease.

Pharmaceutical composition of the present invention comprises Fusaimi derivant or its officinal salt and the acceptable accessories for the treatment of effective dose.

The chemical name of Fusaimi is N-(to the fluoroform phenyl)-2-cyano-3-hydroxy-2-butylene amide, and its derivant has as shown in the formula structure shown in (I):

Wherein, X is H, halogen, C _1-5Alkyl is by OH, NH ₂, the C that replaces of NHR or NRR _1-5Alkyl, or C _1-5Alkoxyl.

The officinal salt of Fusaimi derivant includes but not limited to for example salt of following mineral acid: hydrochloric acid, sulphuric acid, phosphoric acid, pyrophosphoric acid, hydrobromic acid and nitric acid; Perhaps for example following organic acid salt: formic acid, citric acid, malonic acid, maleic acid, fumaric acid, tartaric acid, succinic acid, acetic acid, lactic acid, methanesulfonic acid, p-methyl benzenesulfonic acid, 2-ethylenehydrinsulfonic acid, salicylic acid and stearic acid.Similarly, pharmaceutically useful cation includes but not limited to: sodium, potassium, calcium, aluminum, lithium and ammonium.Those skilled in the art can confirm a variety of nontoxic officinal salts.

The Fusaimi derivant that pharmaceutical composition of the present invention comprised or the treatment effective dose of its officinal salt are every dosage unit 1mg-1g (by Fusaimi).

The acceptable accessories that pharmaceutical composition of the present invention comprised can be pharmaceutical carrier, excipient and other additive that conventional formulation is used.If necessary, pharmaceutical composition of the present invention also can comprise other active component.

Pharmaceutical composition of the present invention can dosage unit preparations oral administration or parenteral administration.Described parenteral administration comprises (for example intravenous), intramuscular or intrathecal injection or infusion etc. in percutaneous, subcutaneous, the blood vessel.

Be suitable for the form that orally uses but tablet, capsule, water or oil suspension dispersed powders or granule, syrup or elixir can be arranged.Can be by any known method preparation in the prior art, such compositions can comprise one or more reagent that is selected from sweeting agent, correctives, coloring agent and antiseptic, so that pharmaceutically attractive in appearance and agreeable to the taste preparation to be provided.

Tablet comprises active component and is suitable for the nontoxic pharmaceutically acceptable excipient of tablet manufacturing.These excipient for example can be inert diluents, as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate, cellulose etc.; Granulating agent and disintegrating agent are as corn starch or alginic acid etc.; Binding agent is as starch, gelatin or arabic gum etc.; Lubricant is as magnesium stearate, stearic acid or Pulvis Talci etc.Tablet can be coating not, perhaps can adopt known technology to carry out coating.This coating can be enteric or delay coating, to postpone disintegrate and the absorption in intestinal, is provided at the slow releasing function in the long time.

Oral formulations can be a hard gelatin capsule, and wherein, active component and inert solid diluent for example calcium carbonate, calcium phosphate or starch are mixed, and perhaps are Perle, and wherein, active component and water or oily medium be Oleum Arachidis hypogaeae semen, liquid paraffin or mixed with olive oil for example.

Water slurry comprises active component and the mixture that is applicable to the excipient for preparing water slurry.This excipient is a suspending agent, as sodium carboxymethyl cellulose, methylcellulose, hydroxypropyl emthylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and Radix Acaciae senegalis; Dispersant or wetting agent can be naturally occurring phospholipid, as lecithin.Water slurry also can comprise antiseptic, coloring agent, correctives and sweeting agent.

Oil suspension can be by active component is suspended in vegetable oil, in Oleum Arachidis hypogaeae semen, olive oil, Oleum sesami or Oleum Cocois, perhaps is suspended in mineral oil such as the liquid paraffin and makes.Oil suspension can comprise thickening agent, as Cera Flava, hard paraffin or spermol.Can add sweeting agent and correctives so that agreeable to the taste oral formulations to be provided.Can add antioxidant in these compositionss such as ascorbic acid carries out anticorrosion.

But dispersed powders is that active component is mixed with dispersant or wetting agent, suspending agent and one or more antiseptic, is suitable for preparing suitable water slurry by adding water.But also can comprise other excipient such as sweeting agent, correctives and coloring agent in the dispersed powders.

Syrup and elixir can be prepared with sweeting agent, as glycerol, propylene glycol, sorbitol, glucose or sucrose.Such preparation also can comprise antiseptic, correctives and coloring agent.

Pharmaceutical composition of the present invention can be through parenterai administration in sterile media.According to excipient that is adopted and concentration, medicine can suspend or be dissolved in the excipient.The injection of parenteral administration can be divided in ampoule, disposable syringe or multiple dose glass or the plastic jar, can be injection or injectable powder.

Pharmaceutical composition of the present invention also can be made cream according to the galenic pharmacy conventional method, absorbs the performance general action for topical application or percutaneous.

Pharmaceutical composition of the present invention is used for the treatment of autoimmune disease, comprises rheumatoid arthritis, systemic lupus erythematosus (sle), lupus nephritis and the nephrotic syndrome, ankylosing spondylitis, psoriasis.

In the above-mentioned lysis of treatment, usually the dosage that adopts is 0.03-5mg/kg/ days, once or divide administration for several times, with the 2-6 month be a course of treatment, generally need to continue 2-4 the course of treatment.

But, be appreciated that, for specific patient, concrete dosage level will depend on multiple factor, comprise the order of severity of the patient's who receives treatment body weight, sex, age, general health situation, meals, administration time, route of administration, drug combination, the disease specific of being treated and disease.

Active ingredient in pharmaceutical Fusaimi derivant of the present invention is the novel immunomodulator of a relative low toxicity, and pair cell immunity and humoral immunization all have inhibitory action.Experimental results show that, the Fusaimi derivant can be controlled non-T cell dependency B cell function effectively, reduce the high-level anti-ds dna antibody of the spontaneous generation of MRL/1pr mice, and control the deposition of immune complex in glomerule of IgG mediation effectively, obviously alleviate brightic pathological manifestations, the pilot system lupus erythematosus there is significantly mitigation, and can alleviates concurrent lymphadenectasis.In the clinical treatment of rheumatoid arthritis, Fusaimi obviously is better than MTX to the improvement of patient's sings and symptoms, and effective percentage and obvious effective rate are all apparently higher than MTX, and untoward reaction (especially gastrointestinal reaction) is starkly lower than MTX.In the treatment of other autoimmune disease, some have produced satisfied effect after the still invalid patient of multiple Drug therapy is with medicine composite for curing of the present invention.

The specific embodiment

Synthesizing of embodiment 1 Fusaimi

Get leflunomide 10g (0.037mol), adding fills in the flask of 2% sodium hydroxide solution 240ml, stirs 1 hour in 23-25 ℃.Add water 1300ml then in reactant liquor, the elimination insoluble matter adds concentrated hydrochloric acid 26ml in colourless filtrate, obtain white depositions immediately, at this moment filtrate pH=1-2.The leaching white solid, (3 * 40ml), 40 ℃ of vacuum concentration get the about 10g of crude product in washing.

With dehydrated alcohol/ethyl acetate (1: 8) 180ml with the crude product recrystallization.The crystallization of separating out gets acicular crystal product 8g (fusing point 230-231.5 ℃) in 40 ℃ of vacuum dryings.With thin layer chromatography (with CH ₂Cl ₂/ HOAc=3/1 is developing solvent) and ¹H-NMR carries out product purity and detects.

The preparation of embodiment 2 capsules

Prescription (1000 capsules):

Fusaimi 1g-1000g

Starch 2.5g-2500g

The Fusaimi raw material is worn into fine powder, cross 100 mesh sieves; With the starch drying, cross 120 mesh sieves; Adopt the equivalent method of progressively increasing to mix supplementary material, cross 120 mesh sieves 2-3 time, make abundant mixing; Inspect qualified back capsule charge by ready samples.

The preparation prescription of embodiment 3 tablets (1000):

Fusaimi 1g-1000g

Lactose 5.0g-5000g

Starch 1.5g-1500g

Pulvis Talci 0.1g-200g

5% starch slurry is an amount of

Low-substituted hydroxypropyl cellulose 0.1g-1000g

OPADRY-OY-GM7305 is an amount of

WHITE

The Fusaimi raw material is worn into fine powder, cross 100 mesh sieves; Adjuvant lactose, starch, L-HPC are crossed 100 orders; Adopt the equivalent method of progressively increasing to mix supplementary material, cross 80 mesh sieves 3-4 time, make abundant mixing; In mixture, add starch slurry system soft material, cross 24 order system wet granulars, dry in 55-60 ℃ of ventilation drying baker; Dried particles is added Pulvis Talci behind 20 order granulate,, make label with the scrobicula stamping of diameter 6.0mm.

OPADRY-OY-GM7305 WHITE being dissolved in 25% ethanol water, stirring 45 minutes, is that 38-45 ℃, whiff pressure are with the label coating under the condition of 3-4kg at the sheet bed tempertaure.Coating warm 10 minutes of (after testing, label weightening finish about 3%) the follow-up continuation of insurance that finishes blows on.

The preparation of embodiment 4 injections

Prescription (1000ml):

Fusaimi 1g-1000g

Sodium chloride 0.1g-900g

Sodium hydroxide 0.01g-200g

Water for injection adds to 1000ml

Sodium chloride is dissolved in 80ml water for injection earlier; Add Fusaimi, it is fully dissolved; Add the 0.1N sodium hydroxide and regulate about pH to 7, add enough water, stirring again, making final volume is 1000ml.Gained solution is sub-packed in the neutral density glass container, with 100 ℃ of sterilizations of flowing steam 30 minutes.

Experimental example 1 Fusaimi is engulfed the influence of chicken red blood cell function to Turnover of Mouse Peritoneal Macrophages

Adopt 50 of male ICR mouses (body weight 19～21 grams), be divided into 5 groups at random, be i.e. matched group, cyclophosphamide group (120mg/kg), three dosage groups of Fusaimi (2,9,45mg/kg), 10 every group.Each organized the mouse stomach administration 5 days, every Mus 0.5ml, and once a day, matched group is given 1% carboxymethylcellulose sodium solution with capacity.In administration the 3rd day, every Mus is lumbar injection 0.25% starch fluid 0.5ml again, after the administration in the 5th day 4 hours, (sterile working is from chicken wing venous blood collection for every Mus lumbar injection 5% chicken erythrocyte suspension 0.5ml, add 5 times of amount A Shi liquid, 4 ℃ of refrigerators are preserved, and use physiological saline solution centrifuge washing three times before using, and are made into 5% chicken red blood cell normal saline suspension).After 30 minutes, the neck execution that only dislocates, lumbar injection HankShi liquid 2.5ml rubs gently by mouse web portion, HankShi liquid is fully rinsed washed peritoneal macrophage.

Then, draw the abdominal cavity washing liquid, drip on two microscope slides, put into the box that is lined with wet gauze, hatched 30 minutes in 37 ℃, incubate completely, the not cell of paster is removed in rinsing in normal saline.Dry, methanol is fixed, Jim Sa Si stain (Giemsa Wright) dyeing, and the distilled water rinsing is dried, and with 100 macrophages of oily mirror counting, calculates percentage rate and phagocytic index, the results are shown in Table 1.

Table 1 Fusaimi is to the influence of Turnover of Mouse Peritoneal Macrophages phagocytic function

The group Mus is counted the phagocytic percentage (phagocytic index of M ± SD) (M ± SD)

Matched group 10 56.1 ± 6.04 1.18 ± 0.20 cyclophosphamide 120mg/kg/d * 5dp.o. 10 34.9 ± 6.97 0.75 ± 0.25 ^*Fusaimi 2mg/kg/g/d * 5dp.o. 10 50.8 ± 8.09 1.08 ± 0.23 Fusaimi 9mg/kg/d * 5dp.o. 10 43.5 ± 10.35 ^*0.87 ± 0.19 ^*Fusaimi 45mg/kg/d * 5dp.o. 10 32.0 ± 5.25 ^{Δ * *}0.64 ± 0.14 ^{Δ * *}Compare with matched group, ^*P＜0.001 ^*P＜0.01; Compare with cyclophosphamide, ^ΔP＞0.05.

As shown in table 1, Fusaimi can obviously suppress the phagocytic function of Turnover of Mouse Peritoneal Macrophages, and middle and high dosage group has been compared significant difference with matched group.

Experimental example 2 Fusaimis are to the mouse cell Immune Effects

(female, male half and half, body weight 23 ± 1g) is divided into 4 groups at random: two dosage groups of Fusaimi (9,45mg/kg), cycli phosphate amine group (120mg/kg) and solvent matched group to adopt 40 of ICR mices.Each lumbar injection 0.2ml medicinal liquid once a day, is put to death mice after continuous 5 days, routinely ³HTdR mixes standard measure and measures T lymphocyte transformation degree.

When the T lymphocyte coagulates element (PHA) In vitro culture with plant, can change into lymphoblast, and vigorous division phenomenon occur.Intracellular protein and nucleic acid are synthetic to be increased, with reflection DNA synthesis rate ³H-thymus pyrimidine nuclear earthenware ( ³HTdR) value of mixing is an index, but quantificational expression lymphocyte transformation degree, thus the lymphocytic immunologic function of reflection T.

Table 2 Fusaimi is to mice ³H-TdR mixes method lymphocyte transformation test result's influence

Group Mus number ³The H-TdR incorporation (CPM ± SD)

Matched group 10 292.9 ± 193.0 cyclophosphamide 120mg/kg/d * 5di.p. 10 140.7 ± 108.9 ^*Fusaimi 9mg/kg/d * 5dp.o. 10 139.1 ± 121.8 ^*Fusaimi 45mg/kg/d * 5dp.o. 10 127.8 ± 99.5 ^*Compare with matched group, ^*P＜0.01.

From table 2 result as seen, the mice of lumbar injection Fusaimi ³The HTdR value of mixing is starkly lower than matched group (P＜0.05), shows that Fusaimi has the obvious suppression effect to the mouse lymphocyte transformation function, promptly suppresses the immunologic function of mouse T cell.

Experimental example 3 Fusaimis produce interleukin 1 (IL-1) test to macrophage

Fusaimi divides five dosage groups, organizes in contrast with lipopolysaccharide (LPS) and cyclophosphamide, is mixed with the solution for standby of the equal capacity of variable concentrations under aseptic condition with RPMI-1640.

Indoor 70 (body weight 18～20g) of healthy mouse inbred lines of getting in the sterile working, be divided into above seven test group at random, every group 10, dislocation is put to death, with 75% soak with ethanol for a moment, it is fixing to lie on the back, and cuts off abdominal skin, injection 5ml HankShi liquid, move abdominal part, reclaim peritoneal fluid, centrifugal (500rpm, 3 minutes) abandoning supernatant, precipitate adds RPMI-1640 to 3ml, centrifugal behind the mixing (2000rpm, 10 minutes), the counting macrophage is diluted to 2 * 10 with 10% calf serum (FCS) cell-preservation liquid (PRMI) RPMI-1640 ⁶/ ml cell number.Add in (1ml hole) in 24 well culture plates, put 37 ℃, 5% CO ₂Cultivate hour, discard non-adherent cell, add the thing that tried of variable concentrations in the macrophage monolayer that obtains, the whole volume in every hole is 1ml, puts 37 ℃, 5% CO ₂Cultivated 48 hours, and collected supernatant (4000rpm, 8 minutes) and filter 20 ℃ of preservations.

Indoorly in the sterile working get 10 of mouse inbred liness in addition, the eyeball blood-letting causes death, 75% soak with ethanol a moment, it is fixing to lie on the back, take out thymus, stripping is surrounding tissue to the greatest extent, tears up thymus to the 10ml graduated centrifuge tube with the method for tearing up, centrifugal (500rpm, 5 minutes) abandoning supernatant, precipitate (thymocyte cell) adds a small amount of RPMI-1640 mixing, adds RPMI-1640 in the homogenate to 70ml, add 210 μ l concanavalin A (ConA) solution, fully mixing.The medicinal liquid that other gets variable concentrations adds to respectively in 96 well culture plates, and every hole 100 μ l add con-A thymus cell suspension 100 μ l (con-A final concentration 3 μ g/ml, thymocyte cell final concentration 1.87 * 10 then ⁸/ ml), put 37 ℃, 5% CO ₂Incubator was cultivated 48 hours.Cultivate and finish preceding 6 hours, every hole adds ³H-TdR 20 μ l (0.2 μ Ci/ hole).Collecting cell is measured radioactivity (cpm) with the LKB automatic liquid scintillation counter on 49 fiber type filter paper.The result represents with the cpm mean value SD of three parts of multiple pipes, stimulates mouse chest cell propagation to mix according to the active record of IL-1 sample when the variable concentrations ³(cpm ± SD) carry out statistical procedures estimates Fusaimi and suppresses the effect that macrophage produces IL-1 the amount of H-TdR.The results are shown in Table 3.Table 3 IL-1 test Fusaimi stimulates mouse chest cell propagation to mix ³H-TdR measures (cpm)

Group sample number cpm ± SD

Fusaimi 20 μ g/ml 10 66.9 ± 52.0 ^*

Fusaimi 15 μ g/ml 10 39.72 ± 13.71 ^*

Fusaimi 10 μ g/ml 10 415.41 ± 497.90 ^*

Fusaimi 5 μ g/ml 10 678.65 ± 293.14 ^*

Fusaimi 1 μ g/ml 10 1263.55 ± 564.32

Lipopolysaccharide (Lps) 1 μ g/ml 10 1101.98 ± 257.0

Cyclophosphamide (cy) 10 μ g/ml 10 1199.3 ± 193.3

Compare with the Lps group, ^*P＜0.01; ^*P＜0.001.

As seen from Table 3, Fusaimi 5,10,15,20 μ g/ml dosage groups produce IL-1 to macrophage the obvious suppression effect, and is the dose dependent relation.

Experimental example 4 Fusaimis are to the inhibitory action of the unidirectional hybrid reaction of human body periphery lymphocyte

The human body peripheral leukocytes is separated: get normal human blood 40ml and put into the 50m test tube, add Ficoll-Hypague (Pharmacia by the bottom, Piscataway, NJ) 10ml, centrifugal 20 minutes in 1600rpm, draw buffy coat, after PBS washing three times, with isolating lymphocyte be suspended in contain 10% deactivation calf serum (FBS, GLBCO) and the RPMI 1644 (GIBCO of 100 μ g/ml green grass or young crops/streptomycins, Grand Lsand, standby in NY).

The T lymphocyte separates: before carrying out the mixed lymphocyte reaction test, leukocyte is suspended in contains 10% deactivation human serum (Diognoctic Inc Meguan is among RPMI1644 WI).With nylon Pilus Caprae seu Ovis post (nylonwool column) and anti-leukocyte 11b antibody (Anti-Leu Ab, Becton Dickinson Mountain View, CA), add complement (Pel-Free, Brown Derr, WL) remove macrophage, B-cell, neutral killer cell, mononuclear cell respectively, 95% is the CD3 positive T cell in the suspension that obtains, and all the other are the B cell.

Mixed lymphocyte reaction (MLR): with the every hole 2 * 10 of isolating T cell ⁵The allogeneic lymphocyte Mixed culture of shining with the ray of raising the price (3000 rad) of equivalent, and add various dose Fusaimi and ciclosporin.The concentration of Fusaimi is respectively 100 μ M, 75 μ M, 50 μ M and 25 μ M, and ciclosporin concentration is 90nM, prepares with cell-preservation liquid.This experiment is carried out with 96 orifice plates at the bottom of the U type, and every dosage group test repeats 5 times (hole).Cultivate after 6 days, in every hole, add 1 μ Ci's ³H labelling thymus pyrimidine detects radioactivity.

Computing formula: % suppresses=[(test specimen radioactivity-background radiation activity)/(contrast specimen radioactivity-background radiation activity)] * 100%.

The results are shown in Table 4.

Table 4 Fusaimi suppresses unidirectional mixed lymphocyte reaction medicine and concentration (%) suppression ratio meansigma methods ± SD

12345 Fusaimis, 100 μ M, 95.85 97.48 86.77 71.27 99.64 90.20 ± 11.6675 μ M, 91.08 77.49 83.69 74.89 93.90 84.21 ± 8.2750 μ M, 54.31 55.08 70.78 58.16 94.15 66.50 ± 16.0225 μ M-17.69-23.82-18.29,31.66 86.00 11.57 ± 47.29 CsA90nM 105.28 104.03 73.53 96.41 47.51 85.35 ± 24.70

Wherein, background is 100% inhibition, does not have any medicine suppression ratio 0%.When Fusaimi concentration was 100M, 75 μ M, 50 μ M, 25 μ M, unidirectional lymphocyte reaction suppression ratio was respectively 90.20 ± 11.66,84.21 ± 8.27,66.50 ± 16.20,11.57 ± 47.29, blue dyeing confirms that lymphocyte does not have destruction, so above concentration is to the lymphocyte free of toxic effects.

Experimental example 5 Fusaimis are to the inhibitory action of the spontaneous system lupus erythematosus of MRL mice

Adopt 10～20 all female MRL/lpr mices in age (providing), body weight 25～30g, 8 every group by U.S.'s Jackson laboratory.The administration group: content is suspended in 1% carboxymethyl cellulose greater than 99% Fusaimi particulate powders, and dosage is 20,35,65mg/kg; And ciclosporin A 50mg/kg.Matched group: equal-volume 1% carboxymethyl cellulose.Each treated animal gavages once through stomach tube every day.Serum 0.3ml is isolated in blood sampling after 63 days.

, cultivated 20 hours in 4 ℃ with the negative contrast of 10 μ g/ml poly glutaminols with anti-double-chain DNA (ds DNA) antibody in the integrated enzyme reaction method detection serum, integrated enzyme reaction method flat board in calf thymus ds DNA 10 μ g/ml.Detect serum and begin from 1: 1000 dilution factor, double dilution is inserted in the integrated enzyme reaction method flat board and is cultivated, and bonded antibody detects through integrated enzyme reaction method detector with anti-mice IgG or the IgM that is associated with horseradish peroxidase, the results are shown in Table 5A, table 5B.

Table 5A Fusaimi (p.o.) is to the influence of MRL/lpr mice ds DNA IgM level

Dilution factor	Matched group	Fusaimi			????CsA50
						????20mg/kg	????35mg/kg	????65mg/kg
		????1000	?1.263±0.167	?0.927±0.153					0.556±0.211 *	0.319±0.057 *	1.297±0.186
????2000	?0.874±0.062				?0.661±0.130	0.342±0.125 *	0.180±0.023 *	0.944±0.152
		????4000	?0.553±0.053	?0.407±0.101					0.215±0.070 *	0.108±0.022 *	0.641±0.119
????8000	?0.287±0.034				?0.200±0.059	0.115±0.035 *	0.050±0.010 *	0.345±0.063
		????16000	?0.137±0.013	?0.087±0.027 *					0.046±0.017 *	0.015±0.006 *	0.180±0.038
????32000	?0.071±0.011				?0.040±0.008 *	0.019±0.011 *	0.003±0.005 *	0.088±0.017
		????64000	?0.065±0.007	?0.027±0.004 *					0.014±0.008 *	0.015±0.007 *	0.053±0.009

^*Compare with matched group, there were significant differences.

Table 5B Fusaimi (p.o.) is to the influence of MRL/lpr mice ds DNA IgG level

Dilution factor	Matched group	Fusaimi			????CsA50
						???20mg/kg	????35mg/kg	????65mg/kg
		????1000	?1.579±0.109	?1.279±0.126					?1.080±0.184 *	0.997±0.095 *	1.809±0.072
????2000	?0.978±0.093				?0.767±0.099	?0.666±0.093 *	0.607±0.045 *	1.281±0.138
		????4000	?0.543±0.050	?0.459±0.049					?0.354±0.048 *	0.312±0.023 *	0.831±0.114
????8000	?0.318±0.048				?0.220±0.025	?0.166±0.024 *	0.175±0.015 *	0.459±0.087
		????16000	?0.186±0.038	?0.130±0.044 *					?0.075±0.009 *	0.107±0.020 *	0.257±0.042
????32000	?0.106±0.013				?0.076±0.038 *	?0.027±0.004 *	0.055±0.018 *	0.154±0.031
		????64000	?0.096±0.009	?0.055±0.028 *					?0.039±0.005 *	0.054±0.014 *	0.101±0.012

^*Compare with matched group, there were significant differences.

After the administration 63 days, animal and anesthesia are put to death down, get all direct-views findable lymph node down, with the electronic scale weighing.The all visible a large amount of enlarged lymph nodes of not treatment group and ciclosporin in treating group are distributed in back, cervical region and intraperitoneal.The lymph node gross weight relatively sees Table 5C.

Table 5C Fusaimi (p.o.) suppresses MRL/lpr mouse lymph knot proliferative effect

The treatment group	Lymph node weight (gram)
		Not treatment contrast	????2.55±0.18
Fusaimi 20mg/kg	????1.90±0.41 *
		Fusaimi 35mg/kg	????0.60±0.13 *
Fusaimi 65mg/kg	????0.35±0.15 *
		????CsA50	????2.12±0.39

^*Compare p＜0.05 with matched group.

Pathological examination results:

Fusaimi 35 and 65mg/kg dosage obviously reverse the inflammation infringement of the glomerule of getting involved to the obvious quantity that reduces the glomerule of getting involved in 9 week of MRL/lpr mice treatment back.Deposition in the IgG immune complex glomerule obviously alleviates, and ciclosporin 50mg/kg and Fusaimi 20mg/kg fail obviously to slow down infringement of glomerule inflammation and IgG deposition.The no significant change of IgM deposition in each group.

Fusaimi 35mg/kg/ days effect, histology and immunohistochemistries to MRL/lpr mice autoimmunity kidney disease:

A) not treatment group shows proliferative glomerulonephritis (glomerule increases, has big amount lymphocyte, neutrophilic infiltration), and has a large amount of IgG to be deposited on the glomerule of getting involved;

B) CsA (50mg/kg/ days) treatment does not obviously alleviate the nephritis pathological changes;

C) Fusaimi treatment group obviously reduces the numbers of glomeruli of getting involved, obviously reverses the glomerule infringement of getting involved, and it is normal to treat Mus mesonephric glomerulus histology in the overwhelming majority.

This experimental example is the result show, Fusaimi can be controlled non-T cell dependency B cell function effectively, reduce the high-level anti-ds dna antibody of the spontaneous generation of MRL/lpr mice, and control the deposition of immune complex in glomerule of IgG mediation effectively, obviously alleviate brightic pathological manifestations, thereby the pilot system lupus erythematosus there is significantly mitigation, and can alleviates concurrent lymphadenectasis.

Treatment example 1 pharmaceutical composition of the present invention is to the curative effect and the safety thereof of rheumatoid arthritis (RA)

Activeness RA patient 190 examples in age 18-65 year (get rid of important organ disease patient, anemia of pregnant woman and women breast-feeding their children, to being subjected to accept golden preparation for treating person before reagent allergy or allergic constitution person, the test) adopt to contrast blind trial at random.Test group 123 examples (male's 14 examples, women's 109 examples), 47 ± 11 years old mean age, average course of disease 69 ± 77 months; Matched group 67 examples (male's 16 examples, women's 51 examples), the equal course of disease of mean aves 50 ± 13 39 ± 50 months.

Test group is taken 2 in Fusaimi 20mg tablet, once a day; 6 of the blank medicines of MTX, 1 time/week.2 of the blank medicines of matched group clothes Fusaimi, 1 time/day; The MTX6 sheet, 1 time/week.Take nonsteroidal medicine Nuo Delun (0.4,1 time/day) after on-test simultaneously, stop using after 4～6 weeks.Once took other patient who does medication slowly before the test one month cleaning phase must be arranged.

The treatment before and take medicine the back 6 weeks, 12 weeks the every index of RA was assessed, try to achieve the average percentage rate that improves.Average improvement rate＜30% is invalid; 〉=30% is effective; 〉=50% is produce effects.The results are shown in Table 6.

Variation before and after the clinical and lab index treatment of table 6 liang group patient (X ± SD)

Fusaimi group MTX organizes project

Treat 12 all rest pains 7.1 ± 2.0-3.0 ± 2.1-4.5 ± 2.3 7.4 ± 1.7-2.4 ± 1.5 of preceding preceding 6 weeks of 12 week treatment in 6 weeks ^*-3.8 ± 2.0 ^*Morning deadlock (minute) 157 ± 256-99 ± 193-119 ± 208 110 ± 65-46 ± 44 ^*-74 ± 68 ^*Grip (kPa) 10.3 ± 6.3 4.6 ± 4.6 7.6 ± 6.9 10.5 ± 6.7 3.6 ± 3.4 ^*6.6 ± 5.4 ^*Articular pain several 15.3 ± 6.3-5.3 ± 4.0-8.8 ± 5.3 13.9 ± 5.2-3.7 ± 2.9 ^*-6.7 ± 4.8 ^*Articular pain index 24.8 ± 14.3-12.9 ± 9.6-17.7 ± 12.5 21.9 ± 12.0-8.1 ± 5.2 ^*-12.1 ± 8.2 ^*Arthroncus several 10.9 ± 5.6-5.4 ± 5.6-7.1 ± 5.9 9.7 ± 4.1-3.0 ± 2.5 ^*-4.6 ± 3.2 ^*Joint swelling index 14.7 ± 9.2-8.1 ± 8.6-10.2 ± 8.8 13.2 ± 6.6-3.9 ± 3.7 ^*-5.8 ± 4.5 ^*Viability 1.6 ± 0.5-0.7 ± 0.4-1.1 ± 0.5 1.5 ± 0.4-0.4 ± 0.3 ^*-0.7 ± 0.4 ^*Patient assessment 7.4 ± 1.9-2.9 ± 1.8-4.4 ± 2.2 7.4 ± 1.7-2.3 ± 1.6 ^*-3.8 ± 2.1 ^*The doctor estimates 7.2 ± 1.9-2.9 ± 1.8-4.3 ± 2.2 7.2 ± 1.7-2.3 ± 1.6 ^*-3.6 ± 2.2 ^*Erythrocyte sedimentation rate (mm/h) 47.6 ± 22.0-20.9 ± 17.4 48.1 ± 24.1-20.7 ± 20.3 CRP (μ g/ml) 30.7 ± 32.8-23.2 ± 28.9 26.5 ± 24.4-17.8 ± 22.5 RF-sxemiquantitative 2.0 ± 0.6-0.6 ± 0.4 1.8 ± 0.6-0.3 ± 0.3 ^*

Annotate: before the treatment be the basic value of every index, is changing value (value-treatment is preceding after the treatment is worth) At All Other Times, and the RF sxemiquantitative is with its logarithm value reciprocal; Two sample means relatively ^*P＜0.05.

As seen from Table 6, Fusaimi group and MTX all can obviously improve patient's clinical symptoms and lab index after 6 weeks of treatment, improve patient's function of joint, but X ray is changed the nothing influence.Except that grip, erythrocyte sedimentation rate, CRP, the Fusaimi group obviously is better than MTX to the improvement of main curative effect index, and its 12 all effective percentage and obvious effective rate are respectively 95.9% and 80.5%, and MTX organizes 12 all effective percentage and obvious effective rate is respectively 85.1% and 62.7%.Two groups of clinical efficacy comparing differences have significance, and (P＜0.05=sees Table 7.

Table 7 liang group patient medication curative effect comparison during 12 weeks

＜30% 30%～50% 〉=50%Effective percentage group example number

The example routine number of the routine number of number (%) (%) (%) (%) Fusaimi group 123 5 (4.1) 19 (15.4) 99 (80.5) (95.9) MTX organizes 67 10 (14.9) 15 (22.4) 42 (62.7) (85.1) ^*Annotate: two groups of curative effects relatively ^*P＜0.05.

In the therapeutic process, untoward reaction takes place in Fusaimi group 20 routine patients, and incidence rate is 16.3%, mainly show as anorexia, nausea, erythra, alopecia, idol has the property a crossed ALT to raise and leucocytes reduction, wherein severe 1 example (erythra), moderate 1 example (ALT rising), slight 18 examples.MTX organizes 27 routine patients untoward reaction takes place, and incidence rate is 40.3%, severe 1 example wherein, and moderate 2 examples, slight 24 examples are significantly higher than Fusaimi group (P＜0.01).All untoward reaction all recover in the course of treatment or after finishing the course of treatment, and 70% recovers in 6 weeks after appearance.

Model case 1

Mrs Xu, woman, 47 years old.Patient's beginning in 87 years extremity size arthralgia, it is particularly evident to swell and ache with two wrists, double knee joint, and two wrists half are tetanic, the obvious obstacle of double knee joint function, paralysis bed 3 years be can't take care of oneself.Erythrocyte sedimentation rate (ESR) 92mm/h before the treatment, rheumatoid factor (RF) 1: 160, C-reactive protein (CRP) 12.1 μ g/ml.The patient once took multiple medicine, as prednisone, MTX, Radix Tripterygii Wilfordii and multiple non-steroidal drug, wherein took behind the Radix Tripterygii Wilfordii in menopause in 38 years old, and accompanied serious alopecia.

The patient begins to take Fusaimi tablet (20mg/ day) in August, 98.Take and second week erythra occurred, reduce by half, week back deflorescence through antianaphylactic treatment and dosage.Again recover former dosage.Treat and can be seated in the 6th week mobilely, little arthralgia is alleviated, and 2～3 meters of row can be stood and hold up to the 12nd week.12 weeks check ESR11mm/h, RF (-), CRP1.91 μ g/ml.Medication 1 year, patient's life can be taken care of oneself.The patient can stair activity, and walking freely.It is all normal repeatedly to check every biochemical indicator therebetween.Existing patient took medicine nearly 3 years, but walking 2 li roads.

Model case 2

Mr. Liu, 54 years old, people from Nanchang.Suffer from RA during the Spring Festival in 1999, in each tame hospital row of Jiangxi Province, western medical treatment, took a large amount of prednisones, " arthralgia aggravated by cold ball ", ant repellent and so in, Western medicine, effect is all undesirable.The patient almost was paralyzed in bed during in February, 2000 to May, can't take care of oneself.Seek medical advice at Shanghai core Ji professor Bao Chunde of hospital place in July, 2000, and multiple medicines is invalid.In December, 2000 rises, and changes the clothes Fusaimi under the Bao professor instructs, and takes just to occur effect in 2nd month.Through the treatment of nearly 1 year Fusaimi, patient's the state of an illness is obviously improved, and not only life can be taken care of oneself, and it is on and off duty also to ride white driving at ordinary times, even can go on business at a distance (Guangzhou, Shenzhen).During the medication, no obvious adverse reaction.

Model case 3

Mrs Ding, 49 years old, Shanghai people from shen village, the RA course of disease 3 years is surplus, is paralyzed in bed, and can't walk, and can not oneself wear the clothes, is button, has a meal, by husband's service.Its husband goes home to she prepares lunch noon every day, and it is influenced to work.Late August calendar year 2001 rises and takes Fusaimi.Onset after 1 month, brothers' arthralgia is clearly better, and the ancon rheumatoid nodules disappears, and erythrocyte sedimentation rate dropped in normal range, and liver function, routine blood test are normal.Medication at present is surplus April, can walk, and life can take care of oneself, and can oneself wear the clothes, cook, and the husband recovers operate as normal.

Treatment example 2 pharmaceutical compositions of the present invention are to the arthritic treatment of psoriasis type

Model case 1. Mries Song, 60 years old, people from Dalian, psoriasis type arthritis.Surplus the psoriasis 20 year, rose in 2000 and the extremities joint infringement to occur, arthroncus, pain, movable unfavorable, the X line is taken the photograph sheet and is shown bilateral carpal joint bone damage, erythrocyte sedimentation rate (ESR): 46mm/ hour.Once used multiple medicines [methotrexate (MTX) etc.] treatment, no produce effects, arthralgia does not have improvement, and erythrocyte sedimentation rate continues to be higher than normally.Take pharmaceutical composition of the present invention from August 16 calendar year 2001.After 2 weeks, erythrocyte sedimentation rate (ESR) was reduced to 30mm/ hour, arthroncus, pain, movable unfavorable improvement.Medication is in the time of 40 days, and the check X-ray film shows that the carpal joint bone damage disappears (unite the consultation of doctors and confirm through radiology department, rheumatism immunity section).In the medication process, no obvious adverse reaction, it is all normal to follow up a case by regular visits to liver function, routine blood test.Patient's ordinary circumstance is good at present, the analgesic of having stopped using, no moving obstacle.

Model case 2. Mr.s Qin, man, 41 years old, people from Nanjing, psoriasis type arthritis.Calendar year 2001 the JIUYUE onset, the little arthroncus of extremity, pain, moving obstacle influences work.Take pharmaceutical composition of the present invention from October calendar year 2001,3 all onsets, arthroncus, pain take a turn for the better, and is easily movable.Take medicine 3 months the time, arthralgia disappears substantially, and is movable accessible, recovers operate as normal.Medication the side effect that times of defecation increases occurs during 6 weeks, and every day 3～5 times, character does not have change, is untreated, and continues medication then, and after 2 weeks, it is normal that times of defecation recovers.There are not other untoward reaction in the medication process.

Treatment example 3 pharmaceutical compositions of the present invention are to the treatment of ankylosing spondylitis

Model case 1. Mr. Wangs, man, 25 years old, people from Shenyang, Liaoning, ankylosing spondylitis.August calendar year 2001 onset, sacroiliac joint pain, the moving obstacle of bending over is taken the photograph sheet through the X line and is diagnosed as ankylosing spondylitis.Make a definite diagnosis after sulfasalazine, prednisone etc. were treated no obvious curative effects 4 months.Rise in January, 2002 and take pharmaceutical composition of the present invention, 6 all onsets, sacroiliac joint swells and ache and alleviates, and the activity of bending over is light.Medication is in the time of 3 months, and swelling and ache disappears substantially, movable accessible.Untoward reaction shows as times of defecation and increases, and every day 3～4 times, character does not have change, occurs during 4 weeks in medication, is untreated, and the back times of defecation recovery of 2 weeks is normal.There are not other untoward reaction during the medication.

Model case 2. Mr. Wangs, man, 49 years old, people from Wuhan, Hubei, ankylosing spondylitis.In January, 1970 onset, regular treatment not.Calendar year 2001 plays the state of an illness and increases the weight of, and bilateral sacrum ilium, knee joint swelling, pain are obvious, and movable obviously obstacles such as bending over, walk influences operate as normal and daily life.Rise August calendar year 2001 and take pharmaceutical composition of the present invention, 2 all onsets, arthroncus, pain take a turn for the better, activity is light.Medication arthroncus in the time of 6 months, pain disappear substantially, and activities such as bending over, walk is accessible, recover orthobiosis and work.Untoward reaction is a skin pruritus, occurs during 5 weeks in medication, adds with the antiallergic agent anti symptom treatment to disappear after 2 weeks.There are not other untoward reaction during the medication.

Claims (7)

1. the pharmaceutical composition of treatment autoimmune disease comprises Fusaimi derivant shown in the formula (I) for the treatment of effective dose

Wherein, X is H, halogen, C _1-5Alkyl is by OH, NH ₂, the C that replaces of NHR or NRR _1-5Alkyl, or C _1-5Alkoxyl, or its officinal salt and acceptable accessories.

2. pharmaceutical composition as claimed in claim 1, wherein said treatment effective dose are every dosage unit 1mg-1g.

3. pharmaceutical composition as claimed in claim 1, described pharmaceutical composition are capsule, and each capsule comprises:

Fusaimi 1mg-1g

Starch 2.5mg-2.5g

4. pharmaceutical composition as claimed in claim 1, described pharmaceutical composition are tablet, and every comprises:

Fusaimi 1mg-1g

Lactose 5mg-5g

Starch 1.5mg-1.5g

Pulvis Talci 0.1mg-0.2g

5% starch slurry is an amount of

Low-substituted hydroxypropyl cellulose 0.1mg-1g

OPADRY-OY-GM7305 is an amount of

WHITE

5. pharmaceutical composition as claimed in claim 1, described pharmaceutical composition are injection, and every dosage unit comprises:

Fusaimi 1mg-1g

Sodium chloride 0.1mg-0.9g

Sodium hydroxide is an amount of

Water for injection adds to 1ml

6. the application of the described pharmaceutical composition of claim 1 in the pharmaceutical preparation of preparation treatment autoimmune disease.

7. application as claimed in claim 6, wherein said autoimmune disease comprises rheumatoid arthritis, systemic lupus erythematosus (sle), lupus nephritis and the nephrotic syndrome, ankylosing spondylitis, psoriasis.